Targeted
cancer nanotherapy
Significant progress has been made in the
development of new agents against cancer and new
delivery technologies. Proteomics and genomics
continue to uncover molecular signatures that are
unique to cancer. Yet, the major challenge remains in
targeting and selectively killing cancer cells while
affecting as few healthy cells as possible.
Nanometer-sized particles have novel optical,
electronic, and structural properties that are not
available from either individual molecules or bulk
solids. When linked with tumor-targeting moieties,
such as tumor-specific ligands or monoclonal
antibodies, these nanoparticles can be used to target
cancer-specific receptors, tumor antigens
(biomarkers), and tumor vasculatures with high
affinity and precision.
Department of Biomedical Engineering,
Emory University and Georgia Institute of Technology,
101 Woodruff Circle Suite 2001,
Atlanta, GA 30322, USA
*E-mail: snie@emory.edu
28 August 2005
by Gloria J. Kim and Shuming Nie*
Conventional cancer therapy and diagnostics involves
the application of catheters, surgery, biopsy,
chemotherapy, and radiation. Most current anticancer
agents do not greatly differentiate between
cancerous and normal cells. This leads to systemic
toxicity and adverse effects. Consequently, the
systemic application of these drugs often causes
severe sideeffects in other tissues (e.g. bone marrow
suppression, cardiomyopathy, and neurotoxicity),
which greatly limits the maximal allowable dose of
the drug. In addition, rapid elimination and
widespread distribution into nontargeted organs and
tissues requires the administration of a drug in large
quantities, which is uneconomical and is often
complicated because of nonspecific toxicity.
Nanotechnology could offer a less invasive alternative,
enhancing the life expectancy and quality of life of the
patient. The diameter of human cells spans 10-20 µm. The size
of cell organelles ranges from a few nanometers to a few
hundred nanometers. Nanoscale devices can readily interact
with biomolecules on the cell surface and within the cells in
a noninvasive manner, leaving the behavior and biochemical
properties of those molecules intact. In their ‘mesoscopic’
size range of 10-100 nm in diameter, nanoparticles have
more surface areas and functional groups that can be linked
to multiple optical, radioisotopic, or magnetic diagnostic and
therapeutic agents. When linked with tumor-targeting
ligands such as monoclonal antibodies, these nanoparticles
can be used to target tumor antigens (biomarkers), as well as
tumor vasculatures with high affinity and specificity. In this
ISSN:1369 7021 © Elsevier Ltd 2005

Glossary
Agonist A drug that triggers an action from a cell or
another drug.
Angiogenesis The process of vascularization of a
tissue involving the development of new capillary blood
vessels. This is caused by the release of chemicals by
the tumor.
Antagonist A compound that inhibits the effect of a
hormone or drug.
Apoptosis A form of programmed cell death induced
by external or internal signals that trigger the activity
of proteolytic caspases, whose actions dismantle the
cell and result in cell death.
Doxorubicin A type of anticancer drug called an
‘anthracycline glycoside’. It works by impairing DNA
synthesis, and hence can target rapidly dividing cells.
Epitope A part of a molecule that an antibody will
recognize and bind to.
Integrin Superfamily of cell surface proteins that are
involved in binding to extracellular matrix components
in some cases.
Liposome Lipid bilayers that form colloidal particles in
an aqueous medium.
Matrix metalloproteinase A member of a group of
enzymes that can break down proteins, such as
collagen, normally found in the spaces between cells
in tissues (i.e. extracellular matrix proteins). They
need Zn or Ca ions to work properly. Matrix
metalloproteinases are involved in wound healing,
angiogenesis, and tumor cell metastasis.
Reticuloendothelial system A system composed of
monocytes and macrophages located in reticular
connective tissue. These cells are responsible for
engulfing (phagocytosis) and removing cellular debris,
old cells, pathogens, and foreign substances from the
bloodstream.
Xenobiotics Chemicals that are foreign to the body.
Xenograft Cells of one species transplanted to another.
RESEARCH REPORT
Passive Targeting EPR Effect
Tumor Environment
Direct Local Delivery
Active Targeting Lectin-carbohydrate Interaction
Ligand-receptor Interaction
Antibody-antigen Interaction
Scheme 1. Targeting strategies for nanoscale drug delivery systems.
article, we discuss different targeting strategies for nanoscale
drug delivery systems (see Scheme 1), and offer a perspective
on cancer nanotherapy.
Passive targeting
Solid tumors have a diffusion-limited maximal size1,2 of
about 2 mm3 and will remain at this size until angiogenesis
occurs, thus granting them access to the circulation3. Rapid
vascularization to serve fast-growing cancerous tissues
inevitably leads to a leaky, defective architecture and
impaired lymphatic drainage. This structure allows an
enhanced permeation and retention (EPR) effect4,5 (first
described by Matsumura et al.6), as a result of which
nanoparticles accumulate at the tumor site. For such a
passive targeting mechanism to work, the size and surface
properties of drug delivery nanoparticles must be controlled
to avoid uptake by the reticuloendothelial system (RES)7. To
maximize circulation times and targeting ability, the optimal
size should be less than 100 nm in diameter and the surface
should be hydrophilic to circumvent clearance by
macrophages (large phagocytic cells of the RES). A
hydrophilic nanoparticle surface safeguards against plasma
protein adsorption, and can be achieved through hydrophilic
polymer coating (e.g. by polyethylene glycol (PEG),
poloxamines, poloxamers, and polysaccharides) or the use of
branched or block copolymers8,9. The covalent linkage of
amphiphilic copolymers (polylactic acid, polycaprolactone,
and polycyanonacrylate) chemically coupled to PEG9-11 is
generally preferred, as it avoids aggregation and ligand
desorption when in contact with blood components7.
An alternative passive targeting strategy is to use the
unique tumor environment in a scheme called tumor-activated
August 2005 29
 RESEARCH REPORT
Biocompatible polymer
Anticancer
agent
Cleavable linkage
responsive to tumor environment
nucleus
Fig. 1 Tumor-activated prodrug delivery and targeting. The anticancer agent is conjugated
to a biocompatible polymer via an ester bond. The linkage is hydrolyzed by cancer-specific
enzymes, or by high or low pH, at the tumor site, at which time the nanoparticle releases
the drug.
prodrug therapy. The drug is conjugated to a tumor-specific
molecule and remains inactive until it reaches the target12
(Fig. 1). Overexpression of the matrix metalloproteinase
(MMP), MMP-2, in melanoma has been shown in a number of
preclinical as well as clinical investigations. Mansour et al.13
reported a water-soluble maleimide derivative of doxorubicin
(DOX) incorporating an MMP-2-specific peptide sequence
(Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln) that binds rapidly and
selectively to the cysteine-34 position of circulating albumin.
The albumin-doxorubicin conjugate is cleaved efficiently and
specifically by MMP-2, releasing a doxorubicin tetrapeptide
(Ile-Ala-Gly-Gln-DOX) and subsequently doxorubicin. pH and
redox potential have also been explored as drug-release
triggers at the tumor site14.
Yet another passive targeting method is the direct local
delivery of anticancer agents to tumors. This approach has
the obvious advantage of excluding the drug from the
systemic circulation. However, administration can be highly
invasive, as it involves injections or surgical procedures. For
some tumors that are difficult to access, such as lung cancers,
the technique is nearly impossible to use.
Active targeting
Active targeting is usually achieved by conjugating to the
nanoparticle a targeting component that provides preferential
accumulation of nanoparticles in the tumor-bearing organ, in
the tumor itself, individual cancer cells, intracellular
organelles, or specific molecules in cancer cells. This approach
is based on specific interactions such as lectin-carbohydrate,
ligand-receptor, and antibody-antigen.
30 August 2005
Lectin-carbohydrate is one of the classic examples for
targeted drug delivery15. Lectins are proteins of
nonimmunological origin that are capable of recognizing and
binding to glycoproteins expressed on cell surfaces. Lectin
interactions with certain carbohydrates are very specific.
Carbohydrate moieties can be used to target drug delivery
systems to lectins (direct lectin targeting), and lectins can be
used as targeting moieties to target cell surface
carbohydrates (reverse lectin targeting). However, drug
delivery systems based on lectin-carbohydrate have been
developed mainly to target whole organs16, which can harm
normal cells. Therefore, in most cases, the targeting moiety is
directed toward specific receptors or antigens expressed on
the plasma membrane or elsewhere at the tumor site.
Multiple drug resistance (MDR), which is a major challenge
in chemotherapy, often stems from the overexpression of the
plasma membrane P-glycoprotein (Pgp)17,18. In general, Pgp
acts as an efflux pump to extrude positively charged
xenobiotics – including some anticancer drugs – out of the
cell. Many tumor cells are resistant to doxorubicin, which is a
Pgp substrate. To overcome the resistance,
poly(cyanocarylate) nanoparticles have been developed19,20.
Adsorption of the nanoparticles onto the plasma membrane
and the subsequent release of doxorubicin leads to saturation
of Pgp. Furthermore, the negatively charged degradation
products of the polymer form an ion pair and neutralize the
positive charge of doxorubicin19, enhancing the diffusion of
the drug across the plasma membrane. Blagosklonny
proposed an approach to selectively kill resistant cancer cells
that is based on a temporary increase in the resistance of
sensitive cells against certain drugs by specific protectors,
such as pharmacological inhibitors of apoptosis21. These
protectors are pumped out by MDR cells, while increasing the
resistance in sensitive cells that do not have active drug
efflux pumps. After applying a cytotoxic drug, sensitive cells
are protected and survive the exposure, while unprotected
MDR counterparts are killed. By abolishing dose-limiting side-
effects of chemotherapy, this strategy might provide a means
to selectively treat aggressive and resistant cancers. Tsuruo
suggested that antibodies to P-glycoprotein overexpressed on
multidrug resistant (MDR) cells could make an attractive
targeting moiety22.
The overexpression of receptors or antigens in many
human cancers lends itself to efficient drug uptake via
receptor-mediated endocytosis (cellular ingestion) – see Fig. 2.
 RESEARCH REPORT

Since glycoproteins cannot remove polymer-drug conjugates Cargo

that have entered the cells via endocytosis23,24, this active Ligand

targeting mechanism provides an alternative route for Cleavable Bond

overcoming MDR. Receptor

The cell surface receptor for folate is inaccessible from the 1. Internalization

circulation to healthy cells because of its location on the apical Cancer Cell Surface
H+
H+
membrane of polarized epithelia, but is overexpressed on the
6. Membrane Fusion
surface of various cancers, including ovary, brain, kidney, H+

breast, and lung malignancies. Surface plasmon resonance

2. Early Endosomes H+
studies reveal that folate-conjugated PEGylated cyanoacrylate H+
5. Recycling Endosomes
nanoparticles have a ten-fold higher affinity for the folate
H+
H+ H+
receptor than free folate11. Folate receptors are often H+

organized in clusters and bind preferably to the multivalent 3. Acidified Endosomes 4. Endosomal Release

forms of the ligand. Furthermore, confocal microscopy

demonstrated selective uptake and endocytosis of folate-
conjugated nanoparticles by tumor cells that bear folate
receptors. Interest in exploiting folate receptor targeting in
cancer therapy and diagnosis has increased rapidly, as attested
by many conjugated systems, including proteins, liposomes,
imaging agents, and neutron activation compounds9,11,25-31.
Tumor targeting by antibodies with engineered properties
(Table 1) is in its infancy, but holds much promise. The
monoclonal antibody (mAb) BR96 (anti-sialyl Lewis Y
antigen), conjugated with doxorubicin, has proved highly
efficacious in tumour xenograft studies32 but, unfortunately,
has shown little or no efficacy in Phase II trials for metastatic
breast cancer33 and advanced gastric adenocarcinoma34,
respectively. Moreover, dose-limiting gastrointestinal
toxicities are observed in the breast cancer trial, because the
immunoconjugate binds to antigen-positive normal cells in
gastric mucosa, small intestine, and pancreas33.
Calicheamicins35-37 and maytansinoids38 are the most
extensively evaluated of many small-molecule toxins that are
used for direct antibody arming, but indirect arming has met
Fig. 2 Nanoparticle drug delivery and targeting using receptor-mediated endocytosis.
with better success. For example, anti-ERBB2
immunoliposomes loaded with doxorubicin show greater
antitumor activity than the free drug or the drug loaded in
nontargeted liposomes in several tumor xenograft
models39,40. Moreover, the systemic toxicity of the
immunoliposome-targeted doxorubicin was much less than
that of free doxorubicin. Bispecific antibodies, which are non-
natural antibodies with two different epitopes, have been
used most widely for the delivery of immune effector cells
and, to a lesser extent, for the delivery of radionuclides,
drugs, and toxins to tumors41,42.
Alternatively, tumor vasculatures can be targeted to allow
targeted delivery to a wide range of tumor types. A number of
angiogenesis inhibitors are undergoing clinical trials (Table 2).
Antiangiogenic therapy prevents neovascularization (the
proliferation of blood vessels in different tissues) by
inhibiting proliferation, migration, and differentiation of
endothelial cells43. Vascular endothelial growth factor (VEGF)
is expressed in many solid tumors. A potent angiogenesis-

Table 1 Antibodies in cancer therapeutics.

Mechanism Antibody target Trade name

Agonist activity CD40, CD137 Various
Antagonist activity CTLA4 MDX-010
Angiogenesis inhibition VEGF Avastin™
Antibody-dependent cell-mediated cytotoxicity CD20 Rituxan®, HuMax-CD20
HER-2/neu Herceptin®
EGF receptor HuMax-EGFr
Toxin-mediated killing CD33 Mylotarg®
Disruption signaling HER-2/neu Pertuzumab (2C4)
Complement-dependent cytotoxicity CD20 Rituxan®, HuMax-CD20
Blockage ligand binding EGF receptor Erbitux™

August 2005 31
 RESEARCH REPORT

Table 2 Angiogenesis inhibitors undergoing clinical trial.

Mechanism Antiangiogenic drug Target Stage of clinical development

Monoclonal antibodies targeting VEGF-A Avastin™ VEGF-A Phase I, II, III
VEGF-Trap VEGF-A Phase I
Antibodies targeting VEGFR-2 IMC-ICII VEGFR-2 Phase I
Receptor tyrosine kinase inhibitors SU5416 VEGFR-2 Phase I, II, III
SU6668 VEGFR-2, bFGFR, PDGFR Phase I, II
ZD 6474 VEGFR-2, EGFR Phase I, II
Endothelial cell proliferation inhibitors ABT-510 Endothelial CD-36 Phase I, II
Angiostatin Various Phase I
Endostatin Various Phase I
TNP-470 Methionine aminopeptidase, Phase I
cyclin dependent kinase 2
Integrin activity inhibitors Vitaxin Integrin ανβ3 Phase I, II
Medi-522 Integrin αvβ3 Phase I
Vascular targeting agents ZD 6126 Endothelial tubulin Phase I
DMXAA Endothelial tubulin Phase I

stimulating protein, it also increases the permeability of
tumor blood vessels. This leads to swelling of the tumor,
ultimately hindering the ability of cancer cells to recruit
blood supply through angiogenesis. VEGF has been used in
liposomes and polymeric nanospheres to deliver angiostatin
and endostatin44,45. The αvβ3 integrin is one of the most
specific biomarkers that can differentiate newly formed
capillaries from their mature counterparts46. Although all
endothelial cells use integrin receptors to attach to the
extraluminal submatrix, one unique receptor (αvβ3 integrin)
is found on the luminal surface of the endothelial cell only
during angiogenesis47. High-affinity αvβ3 selective ligands,
Arg-Gly-Asp (RGD), have been identified by phage display
studies. The cyclic form, which contains a conformationally
constrained RGD, has a higher binding affinity than the linear
form48. Doxorubicin-loaded PEG nanoparticles conjugated to
cyclic RGD49 and paclitaxel-cyclic RGD nanoparticles50 have
nonionizable compounds, micronization, soft-gel technology,
cosolvents, surfactants, or complexing agents have been
used52,34. Since it is faster and more cost effective to
redesign the molecule than to develop a new one, a broadly
based technology applicable to poorly water-soluble drugs
could have a tremendous impact.
Paclitaxel (Taxol™) is a microtubule-stabilizing agent that
promotes tubulin polymerization, disrupting cell division and
causing cell death54. It displays neoplastic activity against
primary epithelial ovarian carcinoma, breast, colon, and lung
cancers. Because it is poorly soluble in aqueous solution, the
formulation that is currently available is Chremophor EL
(polyethoxylated castor oil) and ethanol55. In a new
formulation used in Abraxane (recently approved by the FDA
to treat metastatic breast cancer), paclitaxel was conjugated
Targeting ligand Self-assembled polymer

been reported recently. Anticancer

agent

Covalent bond Tumor specific

Perspective enzyme cleavable linkage

100 nm Receptor-mediated
Nanotechnology is beginning to change the scale and endocytosis

methods of drug delivery. For decades, researchers have been

developing new anticancer agents and new formulations for
delivering existing and new agents.
More than 40% of active substances identified through nucleus

combinatorial screening programs are poorly soluble in Solid tumor

water51. The conventional, and most current, formulations of Receptor nucleus

such drugs are frequently plagued with problems such as poor
and inconsistent bioavailability. The most widely used
method for enhancing solubility is to generate a salt. For
Release of the anticancer agent
Fig. 3 Self-assembled polymeric nanoparticles for both tumor targeting and therapeutic
functions. Inset: delivery of the nanoparticle drugs by receptor-mediated endocytosis and
controlled drug release inside the cytoplasm.

32 August 2005

to albumin nanoparticles56. In addition to circumventing side-
effects of the highly toxic Chremophor EL, which include
hypersensitivity reactions and toxicity to kidney cells and
nerve tissue (nephrotoxicity and neurotoxicity)55,57, the
system cleverly takes advantage of albumin receptors for
improved drug delivery to cancer cells.
For specific targeting, the differences between cancerous
cells and normal cells, which include uncontrolled
proliferation, insensitivity to negative growth regulation, and
antigrowth signals, angiogenesis and metastasis can be
exploited. Thanks to recent advances in proteomics and
genomics, there is a growing body of knowledge of unique
cancer markers. They form the basis of complex interactions
between bioconjugated nanoparticles and cancer cells. Carrier
design and targeting strategies may vary according to the
type, developmental stage, and location of cancer. There is
much synergy between imaging and nanotechnology in
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